Quantitation of uridine and L-dihydroorotic acid in human plasma by LC-MS/MS using a surrogate matrix approach.

Uridine and L-dihydroorotate (DHO) are important intermediates of de novo as well as salvage pathways for the biosynthesis of pyrimidines, which are the building blocks of nucleic acids - DNA and RNA. These metabolites are known to be significant biomarkers of pyrimidine synthesis during the development of DHODH inhibitor drugs for treatment of several cancers and immunological disorders. Here we are reporting a validated LC-MS/MS assay for the quantitation of uridine and DHO in K2EDTA human plasma. Due to presence of endogenous uridine and DHO in the biological matrix, a surrogate matrix approach with bovine serum albumin (BSA) solution was used. Human plasma samples were spiked with stable isotope labeled internal standards, processed by protein precipitation, and analyzed using LC-MS/MS. Parallelism was successfully demonstrated between human plasma (the authentic matrix) and BSA (the surrogate matrix). The linear analytical ranges of the assay were set at 30.0-30,000 ng/mL for uridine and 3.00-3,000 ng/mL for DHO. This validated LC-MS/MS method demonstrated excellent accuracy and precision. The overall accuracy was between 91.9 % and 106 %, and the inter-assay precision (%CV) were less than 4.2 % for uridine in human plasma. The overall accuracy was between 92.8 % and 106 %, and the inter-assay precision (%CV) were less than 7.2 % for DHO in human plasma. Uridine and DHO were found to be stable in human plasma for at least 24 h at room temperature, 579 days when stored at -20 °C, 334 days when stored at -70 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human plasma samples to support clinical studies. Novel Aspect: A surrogate matrix approach to quantify endogenous uridine and DHO concentrations in human plasma.

Integrated risk and recovery monitoring of ecosystem restorations on contaminated sites.

Ecological restorations of contaminated sites balance the human and ecological risks of residual contamination with the benefits of ecological recovery and the return of lost ecological function and ecosystem services. Risk and recovery are interrelated dynamic conditions, changing as remediation and restoration activities progress through implementation into long-term management and ecosystem maturation. Monitoring restoration progress provides data critical to minimizing residual contaminant risk and uncertainty, while measuring ecological advancement toward recovery goals. Effective monitoring plans are designed concurrently with restoration plan development and implementation and are focused on assessing the effectiveness of activities performed in support of restoration goals for the site. Physical, chemical, and biotic measures characterize progress toward desired structural and functional ecosystem components of the goals. Structural metrics, linked to ecosystem functions and services, inform restoration practitioners of work plan modifications or more substantial adaptive management actions necessary to maintain desired recovery. Monitoring frequency, duration, and scale depend on specific attributes and goals of the restoration project. Often tied to restoration milestones, critical assessment of monitoring metrics ensures attainment of risk minimization and ecosystem recovery. Finally, interpretation and communication of monitoring findings inform and engage regulators, other stakeholders, the scientific community, and the public. Because restoration activities will likely cease before full ecosystem recovery, monitoring endpoints should demonstrate risk reduction and a successional trajectory toward the condition established in the restoration goals. A detailed assessment of the completed project's achievements, as well as unrealized objectives, attained through project monitoring, will determine if contaminant risk has been minimized, if injured resources have recovered, and if ecosystem services have been returned. Such retrospective analysis will allow better planning for future restoration goals and strengthen the evidence base for quantifying injuries and damages at other sites in the future.

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion.

The fibronectin fragment, 9th-10th-type III domains (FIII9-10), mediates cell attachment and spreading and is commonly investigated as a bioadhesive interface for implant materials such as titania (TiO2). How the extent of the cell attachment-spreading response is related to the nature of the adsorbed protein layer is largely unknown. Here, the layer thickness and surface fraction of two FIII9-10 mutants (both protonated and deuterated) adsorbed to TiO2 were determined over concentrations used in cell adhesion assays. Unexpectedly, the isotopic forms had different adsorption behaviours. At solution concentrations of 10 mg l(-1), the surface fraction of the less conformationally stable mutant (FIII9'10) was 42% for the deuterated form and 19% for the protonated form (fitted to the same monolayer thickness). Similarly, the surface fraction of the more stable mutant (FIII9'10-H2P) was 34% and 18% for the deuterated and protonated forms, respectively. All proteins showed a transition from monolayer to bilayer between 30 and 100 mg l(-1), with the protein longitudinal orientation moving away from the plane of the TiO2 surface at high concentrations. Baby hamster kidney cells adherent to TiO2 surfaces coated with the proteins (100 mg l(-1)) showed a strong spreading response, irrespective of protein conformational stability. After surface washing, FIII9'10 and FIII9'10-H2P bilayer surface fractions were 30/25% and 42/39% for the lower/upper layers, respectively, implying that the cell spreading response requires only a partial protein surface fraction. Thus, we can use neutron reflectivity to inform the coating process for generating bioadhesive TiO2 surfaces.

Beta-adrenoceptor antagonists affect amyloid nanostructure; amyloid hydrogels as drug delivery vehicles.

Beta-adrenocepetor antagonists (beta-blockers) alter the nanostructure of lysozyme amyloids, resulting in different drug release profiles.

Cross-talk between the insulin-like growth factor (IGF) axis and membrane integrins to regulate cell physiology.

The biology of cross-talk between activated growth factor receptors and cell-surface integrins is an area which has attracted much interest in recent years (Schwartz and Ginsberg, 2002). This review discusses the relationship between the insulin-like growth factor (IGF) axis and cell-surface integrin receptors in the regulation of various aspects of cell physiology. Key to these interactions are signals transmitted between integrins and the IGF-I receptor (IGF-IR) when either or both are bound to their cognate ligands and we will review the current state of knowledge in this area. The IGF axis comprises many molecular components and we will also discuss the potential role of these species in cross-talk with the integrin receptor. With respect to integrin ligands, we will mainly focus on the well-characterized interactions of the two extracellular matrix (ECM) glycoproteins fibronectin (FN) and vitronectin (VN) with cell-surface ligands, and, how this affects activity through the IGF axis. However, we will also highlight the importance of other integrin activation mechanisms and their impact on IGF activity.